Interactions between bacterial pathogens and host innate immunity often determine the outcome of an infection. Toll-like receptors (TLRs) induce antimicrobial mechanisms, but many bacteria have evolved virulence strategies that allow them to evade aspects of this host response. The importance of TLRs for immunity to Salmonella typhimurium has been demonstrated using cells or mice deficient in TLRs or TLR signaling components. A caveat of these studies, however, is that they are typically performed with highly susceptible inbred mouse strains due to a non-functional allele of the Nramp-1 gene. We hypothesize that the extreme susceptibility of Nramp-1 mutant mice may mask host-pathogen interactions between Salmonella and the host innate immune system. Accordingly, we have generated and analyzed TLR-deficient mice with functional Nramp-1. These analyses have resulted in a surprising finding: TLR function is required for Salmonella survival and replication in macrophages and for virulence in mice. In macrophages lacking TLR function, Salmonella fails to induce virulence genes required for intracellular survival and replication. This requirement is based on TLR-dependent acidification of Salmonella containing phagosomes. In this application, we build on these findings and explore the consequences of TLR activation for Salmonella pathogenesis and for the host response. In Aim 1, we will examine whether TLR-dependent acidification and Nramp-1 function cooperate to regulate induction of Salmonella virulence genes. In Aim 2, we will examine in which cell types TLR signaling is required for Salmonella virulence gene induction in vivo. In Aim 3, we will test the hypothesis that Salmonella virulence does not require intracellular replication in TLR-deficient mice. In Aim 4, we will switch to the host side of the host-pathogen interaction and examine why TLR4 plays such a dominant role in the host response to Salmonella infection. PUBLIC HEALTH RELEVANCE: S. typhimurium is the leading cause of bacterial foodborne-disease outbreaks, and human-adapted serovars of Salmonella, such as S. typhi, cause a severe and systemic infection known as typhoid fever. This application addresses the fundamental question of how these bacterial pathogens interact with the innate immune system and how these interactions can influence the outcome of infection.